According to the WHO (World Health Organization), the case where “only a man” has a problem accounts for 24% of the causes of infertility. When the case where both a man and a woman have problems is added, men are involved in about 50% of all the cases. However, the infertility treatment in Japan is placed on the women's side, so that there is currently almost no measure on the men's side.
The causes of male infertility are classified into hypospermia, the case where the volume of semen ejaculated is small, oligozoospermia, the case where the number of sperm is small, and azoospermia, the case where no sperm exist in semen. In addition, the case where the motility of sperm is low is diagnosed as asthenospermia. However, why does the number of sperm decrease and why is the motility rate reduced? These causes can be specified only in a few cases and about 60% of the causes of male infertility remain unclear.
The azoospermia is further divided into two and classified into obstructive azoospermia and non-obstructive azoospermia. Obstructive azoospermia is a pathology in which while sperm are normally formed in the testicles, no sperm come out into ejaculatory semen due to the abnormality of the epididymis and seminal duct. Meanwhile, non-obstructive azoospermia is a pathology in which because of the spermatogenic dysfunction of the testicles themselves, no sperm are formed. For this diagnosis, it is important to check whether or not the blood level of gonadotropin (e.g., FSH, LH), as determined by collecting blood, is abnormal, whether or not the volume and elasticity of the testicles are decreased, and whether or not the vas deferens is unobstructed. The level of serum gonadotropin is an index of spermatogenic function at that time point. The azoospermia having the level of FSH within a normal range means the case where while sperm is generated, none of them come out into ejaculatory semen. Accordingly, the case is diagnosed as obstructive azoospermia. The case where the level of FSH is markedly low or high is diagnosed as non-obstructive azoospermia.
In the non-obstructive azoospermia case where there are sperm verified by testis biopsy, there are many immature or abnormal sperm, the most of which are immotile sperm. Use of such immotile sperm in micro-insemination results in a very low conception rate. Because of this, after the viability of sperm is checked using an HOS test (hypo-osmotic swelling test), micro-insemination is carried out in some facilities (Jeyendran R S, et al., J Reprod Fertil., 1984; 70: 219-28). However, although the HOS test, in which sperm are immersed in a culture solution with a low osmotic pressure, is used to check whether or not the sperm are viable, use of the sperm in micro-insemination is technically difficult. The case where sperm lack motility, not only inside the testicles, but also in ejaculatory semen, has been reported such that the rates of fertilization and embryogenesis by micro-insemination and the conception rate after implantation are extremely low (Vandervorst M, et al., Hum Reprod. 1997; 12: 2429-33). In clinical practice, motile normal-shaped sperm are selected and used for micro-insemination.
In addition, cryopreserved sperm are routinely used in reproductive treatment. However, the freezing treatment reportedly causes a change in the morphology of sperm, the change including damage on mitochondria of sperm; in particular, there is a report in which the freezing treatment largely affects the motility of sperm (O'Connell M, et al., Hum Reprod., 2002; 17: 704-9) (Boitrelle F, et al., J Androl., 2012; 33: 1371-8). There is no reported approach clinically effective in activating sperm having reduced motility subjected to freezing treatment.
Flagellar movement, which is driving force of the motility of sperm, is mediated through motor protein dyneins (Gibbons I R, et al., Science, 1965; 149: 424-426) and tubulins, which constitute a cytoskeletal microtubule serving as a rail (Mohri H, Nature, 1968; 217: 1053-54). The flagellar movement is based on the dynein-mediated sliding of microtubules (Summers K E, et al., Proc. Nat. Acad. Sci. USA, 1971; 68: 3092-3096). The sliding of microtubules causes the bending of a flagellum (Shingyouji, et al., Nature, 1977; 265: 269-270). These phenomena were already uncovered in 1970's. However, the mechanism of flagellar bending at the base and the mechanism of bending wave transmission remain unclear. The flagellar movement is powered by ATP. The ATP that has been synthesized in mitochondria seems to be consumed at each part of a flagellum while the ATP diffuses (Steeghs, et al., Biochem. Biophys. Acta., 1995; 30: 130-138). Meanwhile, there is also a report in which the sperm under an anaerobic condition, for example, in the uterus or oviduct of a mouse or cow, depend on the ATP that has been synthesized in the glycolytic pathway that does not need oxygen (Mukai, et al., Biol. Reprod., 2004; 71: 540-547).
The ATP production in mitochondria needs oxygen. Sperm, one of cells that need oxygen, are known to be affected by reactive oxygen species such as hydrogen peroxide, a superoxide anion, and a hydroxyl radical (Aitken, et al., J. Reprod. Fertil., 1987; 81: 459-469). The superoxide anion, etc., plays a role in making sperm super active and in acquiring fertility, but has different functions depending on, for example, the site or period of action. Meanwhile, seminal plasma contains catalase, superoxide dismutase (SOD), and the like, which can remove reactive oxygen species, so that excessive reactive oxygen species can be removed. Due to this, the sperm, from which seminal plasma has been washed away for use in infertility treatment, are likely to be exposed to reactive oxygen species.
Oxidative stress occurs because reactive oxygen species or free radicals, which have strong oxidation power, are generated in excess. Among them, potent hydroxyl radicals are known to impart various harmful effects on the living body. An approach to eliminating hydroxyl radicals by using an antioxidant and effects thereof have been examined. For example, hydrogen molecules characteristics of selectively reducing and inactivating toxic radicals such as a hydroxyl radical were utilized. This examination involved experimental animal models in which the inhibition of brain ischemia or reperfusion injury and the inhibition of atherosclerosis were exhibited (Non-Patent Document 1). In addition, there are reports on, for example, an improvement in lipid metabolism (Patent Document 1), possible use of a hydrogen-containing agent in the treatment of lung inflammation (Patent Document 2), and a method of inhaling a hydrogen gas for the purpose of aiming at health promotion effects (Patent Document 3). Also, there is a report demonstrating that regarding the motility of sperm, addition of myoinositol to a sperm suspension promotes the recovery of mitochondrial functions, thereby enhancing the motility of sperm (Non-Patent Document 2). This examination, however, is involved in the motile sperm that are ejaculated on the day of collection and can be used for the treatment of in vitro fertilization or micro-insemination. Thus, the above cannot provide a solution to the problem of clinically using immotile sperm. Further, no report has addressed the connection between hydrogen molecules and the motility of sperm.